Feline Enteric Virus Detection Assay

Come J Thieulent, Mariano Carossino, Laura Peak, Udeni B. R. Balasuriya

Published: 2023-10-24 DOI: 10.17504/protocols.io.3byl4qk5jvo5/v1
AI 解读
浏览 1096

Disclaimer

Reference to any commercial materials, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration.

Abstract

The Feline Enteric Virus Detection Assay is intended as an in vitro veterinary reagent set, based on Reverse Transcription quantitative PCR (RT-qPCR), for the detection of feline coronavirus (FCoV)/feline infectious peritonitis (FIP), feline panleukopenia virus (FPV; feline parvovirus), feline rotavirus A (FRV) and SARS-CoV-2 in rectal swabs and fecal samples.

Steps

Reaction Set Up

1.

Thaw all reagents on ice.

2.

Centrifuge all reagents on a benchtop centrifuge to ensure no liquid is in cap and keep on ice

Note
The Feline Enteric Virus Detection Assay does not include an internal control, but positive controls are provided. A positive and a negative control should be run simultaneously with each sample setup.

3.

Setup the Master Mix according to the following table 1:

Table 1. Reaction mix preparation
Table 1. Reaction mix preparation

PROGRAMMING THE THERMOCYCLER

4.

Select the following fluorescence channels: ABYTM, FAMTM, JUNTM and VICTM.

Note
ROXTM should be used as a passive reference dye.

5.

The standard mode should be selected, following the table below:

Table 2. Thermo profile
Table 2. Thermo profile

RESULTS INTERPRETATION

6.

Before analysis of results, the threshold value of each fluorescent dye must be manually set in the region of exponential amplification, typically 0.1 × ΔRn value at the plateau phase.

7.

Each assay is considered valid if the following criteria are met:

Table 3. Positive control validation criteria
Table 3. Positive control validation criteria
8.

The results are qualitative (Positive or Negative). A specimen is considered positive if the Ct value obtained is below the following Ct cut-off values:

Table 4. Ct cut-off values
Table 4. Ct cut-off values

推荐阅读

Nature Protocols
Protocols IO
Current Protocols

Fabrication of angstrom-scale two-dimensional channels for mass transport

Efficient and strand-specific profiling of replicating chromatin with enrichment and sequencing of protein-associated nascent DNA in mammalian cells

The eINTACT method for studying nuclear changes in host plant cells targeted by bacterial effectors in native infection contexts

Rapid reaction optimization by robust and economical quantitative benchtop19F NMR spectroscopy

Profiling native pulmonary basement membrane stiffness using atomic force microscopy

Photoimmunotechnology as a powerful biological tool for molecular-based elimination of target cells and microbes, including bacteria, fungi and viruses

Measuring key human carbohydrate digestive enzyme activities using high-performance anion-exchange chromatography with pulsed amperometric detection

Neural cell isolation from adult macaques for high-throughput analyses and neurosphere cultures

Preparation, validation and use of a vasoactive tryptophan-derived hydroperoxide and relevant control compounds

Neuronal subtype-specific growth cone and soma purification from mammalian CNS via fractionation and fluorescent sorting for subcellular analyses and spatial mapping of local transcriptomes and proteomes

查看全部

扫码咨询